| Staphylococcus aureus(S.aureus)is an important clinical pathogen,which can cause a broad spectrum of infections in humans.Moreover,S.aureus often associates with food poisoning worldwide.Many virulence genes of S.aureus are typically located on mobile genetic elements(MGEs).Horizontal transfer of MGEs among S.aureus strains not only promotes the emergence of“super”bugs,but also accelerates the dissemination of pathogenic strains among animals as well as in humans.Strains from different sources may have various pathogenicity due to the distribution and expression difference of virulence genes.Therefore,it is important to study the genetic background and distribution of virulence genes among the strains from different source,which can help us to understand the spread of pathogenic strain and control the outbreaks of S.aureus associated infectious diseases.Consequently,the main contents and results of this study are as follows:1.S.aureus contamination and MRSA prevalence in raw and processed food commodities of Shanghai City were investigated.A total of607 food samples were randomly collected for S.aureus isolation in Shanghai.Isolation and identification of S.aureus were performed according to GB4789.10-2010.Putative S.aureus isolates were further tested by nuc1-sepecific PCR and 16s RNA sequencing.It was shown that117(19.3%)samples were positive for S.aureus,including 50 raw milk samples,36 fresh meat samples,13 frozen food samples,6 processed soybean products,and 12 vegetables and fruits.A total of 142 different S.aureus isolates were obtained from the 117 S.aureus positive food samples.Then all isolates were further tested for MRSA by the oxacillin disk diffusion method and the mecA gene was also detected by PCR.It was shown that eight isolates(5.6%)harboured the mecA gene and were resistant to oxacillin.Seven of MRSA isolates were from raw milk,the other one from frozen food.2.Multilocus sequence typing(MLST),spa typing and agr typing were used to evaluate the genetic diversity of S.aureus isolates from raw and processed food commodities in Shanghai.It was shown that 26 STs,16 CCs,34 spa types,and 6 agr types were identified among the 142 S.aureus isolates.Among them,four new STs(ST2632-ST2635),four new spa types(t10761,t10777,t10793,and t10798),and one agr I variation were firstly identified.The genotype CC188-t189-agrΙwas the most prevalent,constituting 28.2%of all isolates.It appeared that spa typing had a higher resolution than MLST,and agr types were strongly linked with CCs.3.The prevalence of 25 toxin genes,including 20 enterotoxin genes(sea,seb,sec,sed,see,seg,seh,sei,sej,sek,sel,sem,sen,seo,sep,seq,ser,ses,set and selu),toxic shock syndrome toxin(tsst-1),exfoliative toxin genes(eta,etb and etd),and Panton-Valentine leukocidin genes,was determined in foodborne isolates by specific PCR amplification.The results demonstrated that there were 103(72.5%)isolates carrying at least one toxin gene.Among them,the most frequent toxin gene was sep(43.7%),followed by sej(26.1%)and pvl(21.1%).Furthermore,it was found that the percentage of toxigenic S.aureus isolates was higher for those isolates from meat(80%)or raw milk(89.7%)than any other isolates recovered from processed soybean products(50%),fresh vegetables/fruits(31.3%),or frozen foods(50%).It was determined that the 142 foodborne isolates displayed 52 different toxin gene profiles,with one to 12 toxin genes per isolate in different combinations.Different toxin gene profiles were identified among the same S.aureus lineages.Although no direct association was found between toxin gene profile and the genotype,the isolates belonging to CC5,CC9,CC20,CC50,and CC72 clonal lineages in general carried more toxin genes(>5)compared with the isolates in other CCs.4.egc locus is generally proposed to contribute to the colonization of S.aureus.Moreover,S.aureus infections are generally preceded by commensal colonization.Thus,it is essential to evaluate the virulence characteristic of egc-positive population.In this study,a total of 266 S.aureus isolates,including 142 foodborne isolates and 124 clinical isolates,were obtained in Shanghai.It was shown that a total of 43 isolates(16.17%)were positive for egc locus and there was no significant difference between clinical and foodborne isolates.PCR results for individual egc genes generated seven different egc profiles.It was shown that the percentage of egc profile1(seg,sei,sem,sen,and seo)and egc profile 2(seg,sei,sem,sen,seo,and selu)was 39.53%and 48.54%,respectively.Due to the absence of one or more egc genes,five isolates harbored a unique egc profile,respectively.5.Thirty toxin and twenty adhesin associated genes were detected by PCR in egc-positive isolates.For toxin genes,twenty-six toxin genes were found in egc-positive isolates.Notably,invasive toxin genes showed a high prevalence,such as,76.7%for Panton-Valentine leukocidin encoding genes,27.9%for sec,and 23.3%for tsst-1.Notably,hemolysin encoding genes hla,hlb,hld,and hlg_v were found in all tested isolates.Statistical analysis revealed that only tsst-1 was more significantly prevalent among clinical isolates compared to foodborne isolates.For twenty adhesin associated genes,most were found in most isolates(76.7-100%),while fnbB was only found in 8 egc-positive isolates(18.6%).Overall,high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S.aureus infection.6.These 43 egc-positive isolates were further typed by MLST,PFGE,MLVA and agr typing.It was shown that the 43 egc-positive isolates displayed 17 STs,28 PFGE patterns,29 MLVA types and 4 agr types,respectively.Moreover,MLVA showed a higer resolution than PFGE and MLST.The dominant clonal lineage was CC5-agrII(48.84%).Forty-three egc-positive isolates displayed 33 different toxin gene profiles,with 8 to17 toxin genes per isolate.Among them,26 toxin gene profiles were strain-specific.There was no strong association between toxin gene profile and clonality in most situations.Furthermore,13 adhesion gene profiles were identified in 43 egc-positive isolates.Many isolates belonging to the same clonal lineage had the specific adhesion gene profile.7.By using gentamicin and lysostaphin protection assay,we determined the diversity of Caco-2 cell invasion ability of different S.aureus genotypes.It was shown that strong Caco-2 cell invasion strains were found in CC5,CC9,CC25,CC30,CC50,CC59,CC239 and CC398.In contrast,there was no strong invasion strains in CC1,CC20,CC72 and CC121.It was noteworthy that some isolates belonging to livestock-associated lineage MRSA-CC9 showed strong invasion ability,implying that MRSA-CC9 has a strongly potential to cause disease in humans.In addition,our data demonstrated that biofilm formation and Caco-2 cell invasion ability of strains was positively associated by pearson correlation analysis(R=0.743). |
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